Cupressus Lusitanica Research Articles

Rapid accumulation and metabolism of polyphosphoinositol and its possible role in phytoalexin biosynthesis in yeast elicitor-treated Cupressus lusitanica cell cultures.

Inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)] rapidly accumulates in elicited Cupressus lusitanica Mill. cultured cells by 4- to 5-fold over the control, and then it is metabolized. Correspondingly, phospholipase C (PLC) activity toward phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] is stimulated to high levels by the elicitor and then decreases whereas Ins(1,4,5)P(3) phosphatase activity declines at the beginning of elicitation and increases later. These observations indicate that elicitor-induced biosynthesis and dephosphorylation of Ins(1,4,5)P(3) occur simultaneously and that the Ins(1,4,5)P(3) level may be regulated by both PtdIns(4,5)P(2)-PLC and Ins(1,4,5)P(3) phosphatases. Studies on the properties of PLC and Ins(1,4,5)P(3) phosphatases indicate that PLC activity toward PtdIns(4,5)P(2) was optimal at a lower Ca(2+) concentration than activity toward phosphatidylinositol whereas Ins(1,4,5)P(3) phosphatase activity is inhibited by high Ca(2+) concentration. This suggests that Ins(1,4,5)P(3) biosynthesis and degradation may be regulated by free cytosolic Ca(2+). In addition, a relationship between Ins(1,4,5)P(3) signaling and accumulation of a phytoalexin (beta-thujaplicin) is suggested because inhibition or promotion of Ins(1,4,5)P(3) accumulation by neomycin or LiCl affects elicitor-induced production of beta-thujaplicin. Moreover, ruthenium red inhibits elicitor-induced accumulation of beta-thujaplicin while thapsigargin alone induces beta-thujaplicin accumulation. These results suggest that Ca(2+) released from intracellular calcium stores may mediate elicitor-induced accumulation of beta-thujaplicin via an Ins(1,4,5)P(3) signaling pathway, since it is widely accepted that Ins(1,4,5)P(3) can mobilize Ca(2+) from intracellular stores. This work demonstrates an elicitor-triggered Ins(1,4,5)P(3) turnover, defines its enzymatic basis and regulation, and suggests a role for Ins(1,4,5)P(3) in elicitor-induced phytoalexin accumulation via a Ca(2+) signaling pathway.

Monoterpenes produced by Cupressus lusitanica cultured cells including a novel monoterpene (1S, 2S, 6S)-(+)-1,6-epoxy-4(8)-p-menthen-2-ol

A novel monoterpene, (1S, 2S, 6S)-(+)-1,6-epoxy-4(8)-p-menthen-2-ol, was isolated from an elicitor-treated cell culture of Cupressus lusitanica (Mexican cypress). Ten known monoterpenes - limonene, myrcene, β-ocimene, sabinene, terpinolene, 4-terpineol, α-terpineol, 4(8)-p-menthen-1,2-diol, 4-hydroxyphellandric acid methylester and β-thujaplicin methylether - were also identified. Regioselective metabolisms of monoterpenes were observed in this culture.

Photosynthetically active radiation transmittance of forest plantation canopies in the Ethiopian highlands

Hemispherical photographs and a quantum sensor were used to study the canopy photosynthetic photon flux density (PPFD) transmittance of five forest plantation species in Ethiopia. Red/far-red (R/FR) ratios and temperatures beneath the forest plantations were also measured. The species studied were: Eucalyptus globulus, Pinus patula, Cupressus lusitanica, Grevillea robusta, and Juniperus procera. The canopies of E. globulus, P. patula and G. robusta transmitted about three times as much PPFD as J. procera or C. lusitanica plantations. Furthermore, plantations of G. robusta and E. globulus had the highest, while J. procera had the lowest R/FR ratio. In contrast to J. procera and C. lusitanica, E. globulus and G. robusta had relatively open crowns, higher crown-bases and lower leaf area indices, and as a result, their canopies had higher PPFD transmittance percentages as well as higher below-canopy R/FR ratios and temperatures. From the perspective of understory light regime, plantations of E. globulus and G. robusta are best suited for promoting the natural regeneration of native species. In general, opening of gaps in plantations of heavy-shading tree species may increase the herbaceous layer ground cover, enhance the colonisation and growth of native woody species and consequently may also increase the floristic diversity of mono-specific plantations.

Peroxidases are involved in biosynthesis and biodegradation of β-thujaplicin in fungal elicitor-treated Cupressus lusitanica cell cultures.

• Here, collaboration between peroxidases and H2 O2 in biosynthesis and biodegredation of β-thujaplicin in elicited Cupressus lusitanica was investigated. • The accumulation of a phytoalexin, β-thujaplicin, in C. lusitanica cell cultures can be stimulated by a yeast elicitor. A transient low peak was followed by a high level lasting for 2d, and then a decrease, while peroxidases were activated to a high level just when amounts of β-thujaplicin decreased. In vitro tests revealed that horseradish peroxidase can transform c. 80% of β-thujaplicin in the presence of H2 O2 , and the culture medium was also able to transform β-thujaplicin. • A transient production of H2 O2 occured in the cell cultures immediately after elicitation, following a increase in NAD(P)H-oxidase activity. This H2 O2 production may mediate the elicitor-induced accumulation of β-thujaplicin, because inhibiting H2 O2 production or removing H2 O2 from the cell cultures suppressed elicitor-induced β-thujaplicin accumulation, while exogenously applied H2 O2 or H2 O2 generation system can stimulate β-thujaplicin accumulation. • Both NAD(P)H oxidase inhibitors and peroxidase inhibitors partially inhibited NAD(P)H-dependent O2 - and H2 O2 production. In-gel assay of peroxidase and superoxide anion synthase activity demonstrated that peroxidase isoforms have NAD(P)H-dependent superoxide anion synthase activity. These results suggest that peroxidases can act as superoxide anion synthases to contribute to genecation of H2 O2 that promotes β-thujaplicin production in elicited C. lusitanica cell cultures.

Effect of major inorganic nutrients on β-thujaplicin production in a suspension culture of Cupressus lusitanica cells

The optimum conditions for β-thujaplicin production in a Cupressus lusitanica cell suspension culture were investigated. The conditions required for β-thujaplicin production were clearly different from the conditions for cell growth. The initial phosphate concentration and pH did not affect β-thujaplicin production. A total nitrogen source concentration higher than 3.2 mM suppressed production due to the presence of the ammonium ion. β-Thujaplicin production was observed at 95 mg/l without adding the ammonium ion to the medium. Strict control of major inorganic nutrients was not necessary to produce β-thujaplicin. This finding seems to be favorable for future automated production of β-thujaplicin in commercial cell culture plants.

Multiple signalling pathways mediate fungal elicitor-induced beta-thujaplicin biosynthesis in Cupressus lusitanica cell cultures.

The biosynthesis of a phytoalexin, beta-thujaplicin, in Cupressus lusitanica cell cultures can be stimulated by a yeast elicitor, H(2)O(2), or methyl jasmonate. Lipoxygenase activity was also stimulated by these treatments, suggesting that the oxidative burst and jasmonate pathway may mediate the elicitor-induced accumulation of beta-thujaplicin. The elicitor signalling pathway involved in beta-thujaplicin induction was further investigated using pharmacological and biochemical approaches. Treatment of the cells with calcium ionophore A23187 alone stimulated the production of beta-thujaplicin. A23187 also enhanced the elicitor-induced production of beta-thujaplicin. EGTA, LaCl(3), and verapamil pretreatments partially blocked A23187- or yeast elicitor-induced accumulation of beta-thujaplicin. These results suggest that Ca(2+) influx is required for elicitor-induced production of beta-thujaplicin. Treatment of cell cultures with mastoparan, melittin or cholera toxin alone or in combination with the elicitor stimulated the production of beta-thujaplicin or enhanced the elicitor-induced production of beta-thujaplicin. The G-protein inhibitor suramin inhibited the elicitor-induced production of beta-thujaplicin, suggesting that receptor-coupled G-proteins are likely to be involved in the elicitor-induced biosynthesis of beta-thujaplicin. Indeed, both GTP-binding activity and GTPase activity of the plasma membrane were stimulated by elicitor, and suramin and cholera toxin affected G-protein activities. In addition, all inhibitors of G-proteins and Ca(2+) flux suppressed elicitor-induced increases in lipoxygenase activity whereas activators of G-proteins and the Ca(2+) signalling pathway increased lipoxygenase activity. These observations suggest that Ca(2+) and G-proteins may mediate elicitor signals to the jasmonate pathway, and the jasmonate signalling pathway may then lead to the production of beta-thujaplicin.

Feedback regulation of β-thujaplicin production and formation of its methyl ether in a suspension culture of Cupressus lusitanica

The accumulation of β-thujaplicin in Cupressus lusitanica cell culture was regulated by β-thujaplicin secreted into medium and excess β-thujaplicin was methylated.

New Species of Phoradendron (Viscaceae) from Mexico and Guatemala and a Synopsis of Species in Section Pauciflorae

As presently interpreted Phoradendron section Pauciftorae consists of 15 species. These mistletoes parasitize primarily conifers. We describe seven new species, make status changes for four species, and provide information on the hosts and distribution of all members of the section. New species described are: Phoradendron abietinum Wiens, on Abies durangensis in Chihuahua, Durango, and Jalisco, Mexico; P. acuminatum Wiens, on Cupressus lusitanica in Guatemala; P. flavomarginatum Wiens, on Juniperus flaccida in Nuevo LeOn, Mexico; P. hawksworthii Wiens, on Juniperus in New Mexico, west Texas, and Coahuila, Mexico; P. olivae Wiens, on Cupressus lusitanica in Colima and Jalisco, Mexico; P. rufescens Wiens, on Juniperus spp. in San Luis Potosi, Mexico; and P. sedifolium Wiens on Cupressus lusitanica in Chiapas, and Hidalgo, Mexico. Three taxa previously recognized as subspecies are raised to specific rank: P. densum Torr. ex Trel., P. pauciflorum Torr., and P. libocedri (Engelm.) Howell. Also P. saltillense Trel., which had been placed in synonymy under P. bolleanum subsp. densum, is accorded species status. In addition, three new epiparasitic species of Phoradendron are described. Epiparastic mistletoes are known to parasitize only other species of mistletoes-in this instance Phoradendron or Cladocolea (Loranthaceae).

Native woody species regeneration in exotic tree plantations at Munessa-Shashemene Forest, southern Ethiopia

Regeneration of native woody species was studied in the plantations and the adjacent natural forest at Munessa-Shashemene Forest Project Area, Ethiopia. The aim of the study was to test the hypothesis that tree plantations foster regeneration of native woody species. A total of 60 plots, having 10 × 10 m area each, were studied in monoculture plantations of 4 exotic species (Cupressus lusitanica, Eucalyptus globulus, E. saligna, Pinus patula) and an adjacent natural forest. Ages of the plantations ranged between 9 and 28 years. Soil seed bank analysis was also undertaken from soil samples collected in each of the 60 plots to examine the similarity between the soil seed flora and aboveground vegetation. A total of 56 naturally regenerated woody species were recorded beneath all plantation stands with densities ranging between 2300 and 18650 individuals / ha in different stands. There was a significant difference among plantation stands with regard to understorey density (standard deviation: 4836 ± 1341). Vegetation diversity was assessed through analyses of floristic composition, species richness and abundance. Generally, seedling populations were the most abundant components of the regeneration in most of the plantation stands, forming 68 % of the total regeneration count in all stands. A total of 77 plant species represented by 44 herbs, 13 woody species, 8 grasses and 12 unidentified species were recorded in the soil seed bank from all stands. Similarity between the soil seed bank and aboveground flora was very low implying that the role of soil seed banks is negligible rather dispersal plays an important role in the process of regeneration. These results support the concept that forest plantations can foster the regeneration of native woody species, thereby increasing biological diversity, provided that there are seed sources in the vicinity of the plantations.

Diversity of naturally-regenerated native woody species in forest plantations in the Ethiopian highlands

Woody species diversity and ground layervegetation cover were studied in plantations ofPinus patula, Cupressus lusitanica,Grevillea robusta, and Juniperusprocera, and in surrounding natural forests inWondo Genet, Ethiopia. In the understory of theplantations, a total of 53 naturallyregenerated tree and shrub species belonging to31 families were recorded. Important indigenoustimber species were also represented. Treesaccounted for 72% of all naturally-regeneratedwoody plant species. On the average, 78% ofthe woody species found in plantations werealso found in the natural forest. There was nosignificant variation in understory woodyspecies richness among plantations. C.lusitanica plantations had a significantlyhigher abundance of woody species than those ofP. patula, but the difference wasnot significant in relation to J. procera,G. robusta and the adjacent natural forest.The herbaceous ground cover percentage in G. robusta and P. patula stands wasconsiderably higher than in C. lusitanicaand J. procera stands. Relative toplantations of other species, plantations ofthe native tree species J. procera hadhigher understory species richness, mediumwoody species abundance and low ground cover.Although there was a marked variation inoverstory plantation density, its effect onunderstory species richness and abundance wasnot significant. The density ofnaturally-regenerated woody species inplantations was over three times the usualplanting density in Ethiopia, indicating a highpotential of forest plantations for restoringthe natural forest ecosystems on degraded landsat a comparatively low cost.

Germination of serotinous cone seeds in Cupressus ssp.

Cone serotiny refers to canopy seed storage in cones that remain alive, though closed, for several years. There are some Cupressus species which have only serotinous cones, or both serotinous and non-serotinous cones on the same tree. Scale separation and seed release occurs immediately after the cone ripens in non-serotinous cones, or else the cones remain closed and alive longer, generally up to 15-20 years. Germination tests were done with serotinous cone seeds from C. arizonica Greene, C. benthamii Endl., C. lusitanica Mill., and C. macrocarpa Hartw. Germination was below 50% for all species. The highest levels of germination were obtained from 2- to 7-year-old cones, depending on the species. Cupressus arizonica showed a regular decline in germination values, from 43.5% for seeds from 3-year-old cones to 7.5% for seeds from 7-year-old cones. Cupressus benthamii had a maximum germination value of seeds from 5-year-old cones. Cupressus macrocarpa had a maximum germination capacity of 20.5% for seeds from 6-year-old cones, and it showed 7.5% germination for 12-year-old seeds. Cupressus lusitanica reached 33.5% germination of seeds from 5-year-old cones. Serotinous cones from Cupressus species, usually not collected because it is assumed that their seeds are not viable, can be utilized in cone harvesting, increasing the yield of the cone-bearing trees.

Production of beta-thujaplicin in Cupressus lusitanica suspension cultures fed with organic acids and monoterpenes.

Effects of some organic acids and monoterpenes on production of beta-thujaplicin were studied in Cupressus lusitanica suspension cultures. The fungal elicitor-induced biosynthesis of beta-thujaplicin was promoted by the feedings of malate, pyruvate, fumarate, succinate, and acetate. These results suggest some relationships between acetate/pyruvate metabolism and beta-thujaplicin biosynthesis, or between tricarboxylic acid cycle and beta-thujaplicin biosynthesis. Feedings of C. lusitanica suspension cultures with some monoterpenes inhibited elicitor-triggered beta-thujaplicin biosynthesis, but 2-carene and terpinyl acetate feedings significantly improved the beta-thujaplicin production of C. lusitanica suspension cultures. These results indicate a possible involvement of terpinyl acetate and 2-carene in beta-thujaplicin biosynthesis, as well as potential uses of these monoterpenes in large-scale beta-thujaplicin production.

Biosynthetic Pathway of β-Thujaplicin in the Cupressus lusitanica Cell Culture

Summary The biosynthesis pathway of β-thujaplicin,which has a unique conjugated seven-membered ring structure, was investigated using the Cupressus lusitanica cell culture. We also examined a transformation of the carbon skeleton during its biosynthesis. Incorporation of [10- 14 C] geraniol into β-thujaplicin in the cell culture revealed that this compound was biosynthesized via geranylpyrophosphate (GPP) and hence is a monoterpenoid. The biosynthesis pathway from glucose to GPP and a skeletal rearrangement of GPP to β-thujaplicin are proposed, based on the NMR analysis of β-thujaplicin biosynthesized in C. lusitanica cells from [1- 13 C]-, [2- 13 C]- and [U- 13 C]-glucose. β-Thujaplicin was produced in the C. lusitanica cell from GPP, which was almost solely produced through the GAP/pyruvate pathway; contribution of the classical mevalonate pathway must be negligible, contrary to our previous conclusion. In addition, it was assumed that GPP was transformed to β-thujaplicin via the limonane type skeleton, which is a common intermediate of cyclic monoterpene.

Site index equation for Cupressus lusitanica stands in Munessa forest, Ethiopia

The timber production potential of a given site (site quality) is measured by site index, dominant height of the stand at an index age. Stem analysis data from Cupressus lusitanica stands in Munessa Shashemene Forest, Ethiopia, were used to compute site index equations. The proportional curve method was adopted to construct an average guide curve. Four linear and two widely used non-linear equations were fitted to adjusted mean dominant height–age data. The Schumacher equation was chosen as giving the best fit. Site index curves are reproduced, giving dominant heights in the 12–27 m range at a reference of 15 years.

A study of chemical structure of soft and hardwood and wood polymers by FTIR spectroscopy

Hard and softwood and wood constituent polymers (cellulose and lignin) were studied using Fourier transform infrared (FTIR) spectroscopy. The hollocellulose-to-lignin ratio was estimated for some of the timber species. The structural difference between Klason lignin isolated from softwood (Pinus roxberghii and cupressus lusitanica) and hard wood (Acacia auriculaeformis and Eucalyptus tereticornis) species was studied. © 1999 John Wiley & Sons, Inc. J Appl Polym Sci 71: 1969–1975, 1999

ANATOMÍA DE LA PLÁNTULA DE TRES ESPECIES DE CUPRESSUS (CUPRESSACEAE)

De Magistris, A. A. & Castro, M. A. 1999. Seedling anatomy of three species of Cupressus (Cupressaceae). Darwiniana 37(3-4): 199-207. Structural anatomy of seedlings of Cupressus lusitanica Mill., C. macrocarpa Hartw. and C. sempervirens L. (Cypress) cultivated in Argentina was studied. Seedlings of these species were fixed at eigth different stages of the germination process. Anatomical features of root, hypocotyl, cotyledonary node, cotyledons and epicotyl were described. The root-hypocotyl-cotyledon vascular transition was analized. In the root and in most of the hypocotyl length, the protoxylem exhibits exarch position. At the cotyledonary node the metaxylem differentiates gradual- and laterally divergent from the protoxylem. In sections of the upper zone of the hypocotyl, close to the cotyledonary node, the protoxylem shows an endarch position. In the epicotyl, the undifferentiated procambium alternates with the cotyledonary vascular bundles. The number of xylematic poles is equal to the number of cotyledons in thr three species.

Wood quality of eighteen year old Cupressus lusitanica from Maharashtra

Physical and mechanical properties of 18 year old Cupressus lusitanica, an exotic to India have been reported. The timber was found to be quite knotty and also possessing compression wood. From the data it appears that the timber is weak in strength properties compared to the standard Indian species, Tectona grandis (teak) and Cedrus deodara (deodar) and it can only be used for light packing cases and rural housing.

The effect of abandoned plantations of Pinus patula and Cupressus lusitanica on soilsand regeneration of a tropical montane rain forest in Colombia

Species richness and diversity and soil physical and chemical properties were compared between a mature lower montane rain forest (LMRF) and abandoned 21-year-old plantations of introduced Pinus patula and Cupressus lusitanica in the central Andes of Colombia (2000m). In 0.1ha plots, species richness (number of species) and diversity (Shannon and Simpson indexes) were higher in the LMRF, followed by P. patula and C. lusitanica plantations. There were 56species and 30families of higher plants in the LMRF, 26species and 15families in the P. patula plantation, and 18species and 10families in the C. lusitanica plantation. There were no statistically significant differences (p<0.05) in soil physical or chemical properties between P. patula, C. lusitanica and native LMRF. Nevertheless, soil carbon, the C/N ratio and total bases tended to be higher in the native forest than in the plantations. Available P tended to be higher in the P. patula plantation. These results show that, although abandoned plantations can support some native species, particularly in the understorey, species richness is far from that of the native forest.

Variation and inheritance of resistance to cypress aphid, Cinara cupressi Buckton in Cupressus lusitanica Miller

Summary. Cupressus lusitanica seedlings from open‐pollinated seeds of 18 families were inoculated with day‐old first instar Cinara cupressi. Aphid survival was used to determine the genetic basis and inheritance of resistance to the insect. There was marked variation in aphid survival both between and within families. An individual‐tree narrow‐sense heritability of 0.76 ± 0.30 shows strong additive genetic control which could allow effective selection and breeding for resistance. Resistant parents produced resistant progeny while susceptible parents produced susceptible progeny. There were notable exceptions as some susceptible parents produced highly resistant progeny, indicating that they had acquired pollen from resistant neighbours. Recovery of aphid damaged trees is evident implying that care should be taken in selecting for resistance. The strong additive variance and potentially high heritability indicate that one cycle of selection may yield a resistant population, while intraspecific crossing may give better results. Implications of the results in a C. lusitanica breeding programme are discussed.

Improved Culture Conditions for the Production of .BETA.-Thujaplicin by Suspension Cell Cultures of Cupressus lusitanica.

An improvement in the production of β-thujaplicin (hinokitiol) by the Cupressus lusitanica suspension cell cultures is described. For cell growth, a sufficient supply of nutrients was necessary but Fe (II) had an inhibitory effect. The cells grew well in a modified Gamborg B5 medium (IS-1) containing Fe of 0.01mM which is one tenth of that in the original B5 medium. On the contrary, insufficient inorganic nutrients and excess Fe were effective for the β-thujaplicin production. Thus, the cells accumulated 22mg of β-thujaplicin/g dry cell weight in 3 days after elicitation, when 20g/l glucose was used as a carbon source in the other modified Gamborg B5 medium (IS-2) which contained 0.25mM Fe and major nutrients at 0.1 strength of the original B5 medium. The glucose dose can be greatly reduced without seriously affecting the β-thujaplicin production level, if a proper osmotic pressure regulator is present in the medium.